Serious obstacles to commercially viable processes for the enzymatic resolution of enantiomeric mixtures of hydrophobic esters exist. For example, when using an enzymatic conversion process in the presence of an organic solvent, the rate of enzyme inactivation is very high relative to the same process performed in an aqueous solvent. A confounding problem is that solvents which are less destructive to the catalyst are often less able to solubilize the more hydrophobic substrates. Ideally, many processes would be more efficient if they were performed in more hydrophobic solvents, such as non-miscible organic solvents. One goal of the present invention is to provide a non-homogenous system, which allows higher concentrations of hydrophobic substrates to be converted to product, while simultaneously consuming less catalyst.
The above-cited obstacles must be overcome in order to reduce the cost of producing enantiomeric drugs anti-viral drugs. Such drugs are vital towards winning the struggle to conquering emerging viral diseases. For example, even today, the rate of HIV infection continues at a staggering pace, with 16,000 new infections per day worldwide [Balter, M. Science 280, 1863–1864 (1998)]. There are areas of sub-Saharan Africa where at least 25% of the population are infected, for example in Botswana and Zimbabwe. The cost of anti-viral drugs, however, is currently far beyond the reach of most such victims of HIV infection.
Nucleoside analogues, such as 3′-thiaribofuranonsyl-βL-cytosine (“3-TC”), 3′-azido-3′-deoxythymidine (AZT) [Blair E., Darby, G., Gough, E., Littler, D., Rowlands, D., Tisdale, M. Antiviral Therapy, BIOS Scientific Publishers Limited, 1998], (−)-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (“FTC”) and 2′,3′-dideoxy-3′-thiacytidine are important antiviral agents [Liotta, D. C. 216th ACS National Meeting, Medicinal Chemistry Abstract, Boston, Mass., August 2327, 1998; Hoong, L. K., Strange, L. E., Liotta, D. C., Koszalka, G. W., Burns, C. L., and Schinazi, R. F., J. Org. Chem. 1992, 57, 5563-5565]. 3-TC has been marketed as both an anti-HIV and an anti-HBV drug and FTC is under clinical trial for evaluation as an anti-viral drug [Liotta, D. C., Schinazi, R. F., and Choi, W.-B., U.S. Pat. Nos. 5,210,085, 5,700,937 and 5,814,639]. Since it is the (−) enantiomer of both (−)-FTC and (−)-2′,3′-dideoxy-3′-thiacytidine, which exhibits the most potent anti-viral activity and the least toxicity, as compared to the corresponding (+)-isomers, there is a pressing need for efficient cost-effective methods of preparation of both the (−)-FTC and (−)-2′,3′-dideoxy-3′-thiacytidine isomers to expand treatment options of patients throughout the world [Liotta, D. C. 216th ACS National Meeting, Medicinal Chemistry Abstract, Boston, Mass., Aug. 23–27, 1998, Hoong, L. K., Strange, L. E., Liotta, D. C., Koszalka, G. W., Burns, C. L., and Schinazi, R. F., J. Org. Chem. 1992, 57, 5563-5565].
Many hydrolase enzymes have been used for the resolution of FTC esters [Hoong, L. K., Strange, L. E., Liotta, D. C., Koszalka, G. W., Burns, C. L., and Schinazi, R. F., J. Org. Chem. 1992, 57, 5563–5565]. Impediments remain, however, to developing practical enzyme mediated chemical processes for the production of FTC and similar compounds. First, the solubility of many FTC esters in aqueous media is too low to achieve economically viable production of resolved product. One possible solution has been to add a water miscible co-organic solvent to increase the concentration of the ester in solution. An example is the use of solutions of acetonitrile and water [Hoong, L. K., Strange, L. E., Liotta, D. C., Koszalka, G. W., Burns, C. L., and Schinazi, R. F., J. Org. Chem. 1992, 57, 5563–5565; Liotta et al., U.S. Pat. No. 5,827,727]. Although the use of a water miscible organic solvent and water solution increases the concentration of substrate in solution, it has the unfortunate effect of drastically lowering the enzyme catalyzed conversion and enzyme stability. This problem is especially pronounced, where the substrate is not completely dissolved, but is also present as an undissolved solid suspension (high concentration of substrate loading). Similar results were obtained in our laboratory. When water miscible organic solvents, such as isopropanol, dimethylformamide (DMF), 1-methyl-2-pyrrolidinone, dimethylsulfoxide (DMSO), methanol, acetonitrile, ethanol, 1-propanol were used as co-solvent for the resolution, the maximal substrate concentration loading was 3%. The presence of undissolved substrate decreased the enantioselectivity when the substrate concentration was beyond 3%. Furthermore, use of a water miscible organic solvent and water solution, at concentrations of water miscible organic co-solvents of greater than 20%, had a pronounced negative impact on enzyme activity, especially for porcine liver esterase (PLE).
The present invention specifically addresses several obstacles in the art that had the effect of making enzymatic resolution of enantiomeric mixtures uneconomical. First, it was thought that enzymatic conversion should be performed under homogenous conditions, because biphasic systems result in poor reproducibility [See Liotta et al., U.S. Pat. Nos. 5,827,727, 5,892,025, 5,914,331]. One potential advantage for the use of non-homogenous systems would be in enhanced solubilization of the substrate. Presumably, in a non-homogenous system, a higher concentration of many hydrophobic substrates could be accommodated. Prior to the present invention, it was believed that alcohol solvents should be avoided, because these solvents denature enzymes [Liotta et al., U.S. Pat. Nos. 5,827,727, 5,892,025, 5,914,331]. The present invention is an advance over the art because it specifically provides for the use of alcohol solvents which form non-homogenous systems with water. In addition, the use of non-homogenous solvent systems provides increased solubilization of more hydrophobic substrates than could be accomodated previously in the art. Furthermore, the present invention discloses a process which requires less enzyme per unit of product.
Additional improvements achieved via the present invention permit the use of several alcohol solvents in an enzymatic process. In addition, the present invention provides an alternative process node, wherein enzyme and organic solvent requirements are further reduced by the addition of surfactants. Finally, the present invention is directed to providing a more efficient enzymatic process which maintains the enantioselectivity at a high level.